cell cyle regulation in yeast

8/7/2019 cell cyle regulation in yeast

1/23

10 Yeast Growth and the Cell Cycle10.1 Vegetative Reproduction in Yeast

Mitotic division is initiated when cells attain a critical cell size and are stimulated by exogenous or

endogenous signals during interphase. A prerequisite is that DNA synthesis guarantees proper

duplication of the genetic material and that all substrates critical to anabolic pathways are available. In

mammalian cells, the mitotic phase can be subdivided into a number of steps that are characterized by

particular states of orienting the spindle pole bodies, sorting and movement of the duplicated

chromosomes, and finally, cell separation after cytokinesis (Figure 10-1). While all processes pertinent

to the cell cycle have been highly conserved among all eukaryotes, ascomycetous fungi, and in

particular S.cerevisiae, exhibit several peculiarities with respect to cell division and cytokinesis

[Bouquin et al., 2000; Bidlingmaier et al., 2001].

Figure 10-1: Phases in the mitotic cycle.10.1.1 Yeast BuddingBudding is the most common mode of vegetative growth in yeasts and multilateral budding is a

typical reproductive characteristic of ascomycetous yeasts, including S. cerevisiae. Yeast buds are

initiated when mother cells attain a critical cell size at a time coinciding with the onset of DNA

synthesis. This is followed by localized weakening of the cell wall and this, together with tension

exerted by turgor pressure, allows extrusion of cytoplasm into an area bounded by new cell wall

material. The regulation of particular cell wall synthetic enzymes and transport of specific bud plasma

membrane receptors are key steps in the emergence of a bud. Chitin forms a ring at the junction

between the mother cell and the newly emerging bud to finally result in the generation of a daughter

cell. After cell separation, this ring will be retained at the surface of the mother cell and form the so-

called bud scar (Figures 10-2 and 10-3), and a birth scar at the surface of the daughter. The number of

bud scars left on the surface of a yeast cell is a useful determinant of cellular age.


2/23

Several recent papers have been devoted to budding in yeast [Roemer et al., 1996; Barral et al., 1999;

Barrett et al., 2000; Manning et al., 1999; Ni & Snyder, 2001; Sheu et al., 2000; Swaroop et al., 2000;

Vogel et al., 2000].

Figure 10-2: Budding yeast cell.

Figure 10-3: Yeast bud and bud scar.

Table 10-1: Examples of components important for budding

Genes Gene product characteristics Mutant phenotypes

General bud-site selection Random budding in haploids and diploidsBUD1/RSR1 Ras-related protein

BUD2 GTPase-activating protein (GAP)BUD5 GDP-GTP exchange factor (GEF)

Axial bud-site selection Bipolar budding in haploidsBUD3 Novel

BUD4 GTP_binding domainAKL1 a-factor protease

BUD10/AXL2 Type-I plasma membrane glycoprotein

Polarity establishment Round, multinucleate cells unable to bud

CDC24 GEF for Cdc42pCDC42 Rho/Rac GTPase

BEM1 SH3 domains

Diploid bud-site selection Random budding in diploids/mother cells

ACT1 Actin

SPA2 Coiled-coil domainRSV161, RSV167 SH3 domainBNI1, BUD6, BUD7 ?

BUD8 ?BUD9 ?

Septin-ring Bipolar budding in haploidsCDC3, CDC10, CDC11, CDC12 10-nm filament ring components

GTP-binding domain


3/23

During bud formation, only the bud but not the mother cell will grow. Once mitosis is complete and the

bud nucleus and other organelles have migrated into the bud, cytokinesis commences and a septum is

formed in the isthmus between mother and daughter. A ring of proteins, called septins (Figure 10-4),

are involved in positioning cell division in that they define the cleavage plain which bisects the spindle

axis at cytokinesis. These septins encircle the neck between mother and daughter for the duration of

the cell cycle.In S. cerevisiae, cell size at division is asymmetrical with buds being smaller than mother cells when

they separate. Also cell division cycle times are different, because daughter cells need time (in G1

phase) to attain the critical cell size before they are prepared to bud.Budding is not a randomized, uncontrolled process; cellular geometry is explicitly important in

localizing budding sites. Numerous studies have endeavoured to explain at the cellular and molecular

level how polarized cell growth is regulated and how the site of emerging buds is chosen. Bud site

selection depends on several physiological and genetic factors. For example, cell mating type is

important, and a and haploid cells are kwon to exhibit an axial budding pattern, where as

a/ diploids exhibit a bipolar budding pattern. Axial budding means that mother and daughter cells

form a new bud near the preceding bud scar and birth scar, respectively. Bipolar budding is when

daughter cells bud firstly away from their mother, while mother cells either bud away or toward

daughter cells.

The hierarchy of cell polarity is governed by the interplay of various genes that dictate the orientation

of cytoskeletal elements. For example, the bud-site selection genes (BUD genes) are required for

determining the orientation of actin fibres, and genes for bud formation (such as CDC24, CDC42,

BEM1) direct cell surface growth to the developing bud. Budding is strictly connected to events in the

cell cycle (see below) in that cyclins and cyclin-dependent kinases play a decisive role in actin

assembly and in localizing and timing of bud emergence.

It may be mentioned briefly that fission yeasts, like Schizosaccharomyces pombe, divide exclusively

by forming a cell septum analogous to the mammalian cell cleavage furrow, which constricts the cell

into two equal-sized daughters.

10.1.2 Yeast Septins

Septins are highly conserved cytoskeletal elements found in fungi, mammals, and all eukaryotes

examined thus far, with the exception of plants [Barral et al., 2000; Casamayor & Snyder, 2004]. The

septin proteins assemble into filaments that lie underneath the plasma membrane. In Saccharomyces

cerevisiae, where they were first identified, septins are visible as electron-dense cortical rings at the

mother bud neck. In multicellular organisms, they are found at the cleavage furrow and other cortical

locations. Consistent with their localization, septins have been shown to be required for cytokinesis in

yeast, Drosophila melanogaster, and mammalian cells.

Recent evidence in yeast has demonstrated that septins participate in a variety of other cellular

processes, including cell morphogenesis, bud site selection, chitin deposition, cell cycle regulation, cell

compartmentalization, and spore wall formation. Since septins participate in many cellular processes,

it is not surprising that a diverse set of proteins have been found associated with the yeast septin

cytoskeleton.


4/23

The septins have a highly conserved structure. They contain a central GTP-binding domain flanked by

a basic region at the amino terminus, and most septins contain a coiled-coil domain at the carboxy

terminus. In yeast, five septins, Cdc3, Cdc10, Cdc11, Cdc12, and Shs1, localize to the mother bud

neck in vegetatively growing cells. Cdc3 and Cdc12 are essential for growth at all temperatures,

whereas Cdc10 and Cdc11 are required only at elevated temperatures. Shs1 is a nonessential septin.

Cells containing temperature- sensitive mutations in either CDC3, CDC10, CDC11, or CDC12delay at

a G2 checkpoint and arrest at the restrictive temperature, forming extensive chains of highly elongated

cells.

Figure 10-4: Functions of septins.Table 10-2: Diversity of septin expression and function.

Gene Function Localization Biochemistry

CDC3CDC10CDC11CDC12

Essential for cytokinesis andpolar-bud growth control. Cdc3pand Cdc12p required for viability,but not Cdc10p and Cdc11pinsome backgrounds. Reqired forproper regulation of the Gin4p andHsl1p kinases

Bud neck, site ofbud emergence,base of schmoo

Found in 370 kDa complexthat can form filaments invitro

SEP7 Required in vivo for properregulation of Gin4p kinase

Bud neck, site ofbud emergence

Can complex with Cdc3p,Cdc10, Cdc11p, Cdc12p

SPR3 Sporulation efficiency Prospore wall No data

SPR28 No obvious phentype Prospore wall No data

Table 10-3: Budding yeast septin-protein interations.

Protein Function Septin-dependentlocalization

Genetic/physicalinteractions

Localization

Gin4pHsl1Kcc4

Protein kinases that function inseptin localization and cell cycleprogression

Yes Gin4pinteractsphysically andgenetically

with septins

Bud neck, site of budemergence

Bni4p Reqired for normal chitindepositionand morphology

Yes Two-hybridinteractionwith Cdc10pand Chs4p

Bud neck


5/23

Chs3pChs4p

Required for normal chitinsynthesis

Yes SyntheticlethalinteractionbetweenChs4p andCdc12p


Yck1pYck2p

Casein kinase I homologs,required for septin localization,

cytokinesis, morphogenesis andendocytosis

No Unknown Bud neck, sites ofpolarized growth,

plasma membrane

Bud3pBud4pSpa2p

Bud site selection YesYes??

UnknownUnknownSyntheticlethalinteractionwith Cdc10p


Bni1p Cytokinesis/morphogenesis ?? Syntheticlethalinteractionwith Cdc12p

Bud tip

Myo1p Type II myosin. Plays a role incytogenesis

Yes Unknown Bud neck

Arf1p Morphogenesis during mating ?? Two-hybridinteraction

with Cdc12p

Base of matingprojections

10.1.3 Yeast Spindle Pole Body (SPB)Nucleation of microtubules by eukaryotic microtubule organizing centers (MTOCs) is required for a

variety of functions, including chromosome segregation during mitosis and meiosis, cytokinesis,

fertilization, cellular morphogenesis, cell motility, and intracellular trafficking. Analysis of MTOCs from

different organisms shows that the structure of these organelles is widely varied even though they all

share the function of microtubule nucleation. Despite their morphological diversity, many componentsand regulators of MTOCs, as well as principles in their assembly, seem to be conserved [Segal et al.,

2001; Jasperson et al., 2004; .Cheeseman & Desay, 2004].

.

The spindle pole body (SPB) is the sole site of microtubule organization in the budding yeast

Saccharomyces cerevisiae. SPBs are embedded in the nuclear envelope throughout the yeast life

cycle and are therefore able to nucleate both nuclear and cytoplasmic microtubules. The small size of

the yeast SPB, its location in a membrane, and the fact that nearly all genes involved in SPB function

are essential have presented significant challenges in its analysis. Nevertheless, the SPB is perhaps

the best-characterized microtubule organizing center (MTOC).

The SPB is a cylindrical organelle that appears to consist of three disks or plaques of darkly staining

material (Figure 10-5): an outer plaque that faces the cytoplasm and is associated with cytoplasmic

microtubules, an inner plaque that faces the nucleoplasm and is associated with nuclear microtubules

and a central plaque that spans the nuclear membrane. One side of the central plaque is associated

with an electron-dense region of the nuclear envelope termed the half-bridge. This is the site of new

SPB assembly because darkly staining material similar in structure to the SPB accumulates on its

distal, cytoplasmic tip during G1 phase of the cell cycle.

Careful analysis of SPB size and structure indicates that the SPB is a dynamic organelle. In haploid

cells, the SPB grows in diameter from 80 nm in G1 to 110 nm in mitosis. The molecular mass of adiploid SPB, including microtubules and microtubule associated proteins, is estimated to be 11.5

GDa. However, only 17 components of the mitotic SPB have been identified to date (Table 10-4).


6/23

Table 10-4: Yeast spindle pole body components.

Protein SPB location Role in SPB function

Tub4 -tubulin complex MT nucleation

Spc98 -tubulin complex MT nucleation

Spc97 -tubulin complex MT nucleation

Spc72 OP, HB -tubulin binding protein

Nud1 OP, satellite MEN signallingCnm67 IL1, OP, satellite Spacer, anchors OP to CPSpc42 IL2, OP, satellite Structural SPB core

Spc29 CP, satellite Structural SPB core

Cmd1 CP Structural Spc110 binding proteinSpc110 CP to IP Spacer,-tubulin binding proteinNdc1 SPB periphery Membrane protein, SPB insertion

Mps2 SPB periphery Membrane protein, SPB insertionBbp1 SPB periphery SPB core, HB linker to membrane

Kar1 HB Membrane protein, SPB duplicationMps3 HB Membrane protein, SPB duplication

Cdc31 HB SPB duplicationSfi1 HB SPB duplication

Mpc54 MP Replace Spc72 in meosis I

Spo21 MP Replace Spc72 in meosis II

CP= Central plaque; HB=half-bridge; IL1=Inner layer 1; IL2= Inner layer 2; IP=Inner plaque;MEN= ; cMT= Cytoplasmic MT; nMT= Nuclear MT; OP=Outer plaque.

Figure 10-5: Location of protein components of the spindle pole body.


7/23

Regulators of SPB duplication and function associate with the SPB during all or part of the cell cycle.

Mps1, a conserved protein kinase required for multiple steps in SPB duplication and also for the

spindle checkpoint, localizes to SPBs and to kinetochores

Figure 10-6: SPB duplication pathway.

SPB duplication (Figure 10-6) can be divided into three steps: (1) half-bridge elongation and

deposition of satellite material, (2) expansion of the satellite into a duplication plaque and retraction of

the half-bridge, and (3) insertion of the duplication plaque into the nuclear envelope and assembly of

the inner plaque. Following completion of SPB duplication, the bridge connecting the side-by-side

SPBs is severed, and SPBs move to opposite sides of the nuclear envelope (4). The requirements for

various gene products in each step are shown in the figure. SPC72, NUD1, and CNM67are probably

required for step 2. SPBs are not synthesized de novo. Consequently, every time a cell divides it must

duplicate its SPB, as well as its genome, to ensure that both the mother and daughter cell contain one

copy of all 16 chromosomes and one SPB. SPB duplication occurs in G1 phase of the cell cycle;

however, defects in SPB duplication are not detected until mitosis when cells fail to form a functional

bipolar spindle. Generally, SPB defects cannot be reversed at this point, so cells will eventually

attempt chromosome segregation with a monopolar spindle, which results in progeny with aberrant

DNA content and/or SPB number. Therefore, accurate SPB duplication during G1 is essential to

maintain genomic stability.


8/23

10.2 The Yeast Cell Cycle

10.2.1 General

The cell cycle can be defined as the period between division of a mother cell and subsequent divisionof its daughter progeny. The regulatory mechanisms that order and coordinate the progress of the cell

cycle have been intensely studied [Mal & Nurse, 1998; Futcher, 2000;.Lauren et al., 2001]. Numerous

proteins that have been characterized through mutations are collectively designated as cell division

cycle (Cdc) proteins.

The eukaryotic cell cycle involves both continuous events (cell growth) and periodic events (DNA

synthesis and mitosis). Commencement and progression of these events can formally been

distinguished into pathways for DNA synthesis and nuclear division, spindle formation, bud emergence

and nuclear migration, and cytokinesis. However, from a molecular viewpoint these processes are

intimately coupled (Figure 10-6).

Figure 10-7: Cell cycle phases and physiological processes.

The periodic events can be divided into four phases (Figure 10-7): DNA synthesis (S phase); a post-

synthetic gap (G2 phase); mitosis (M phase); and a pre-synthetic gap (G1 phase). For division, yeast

cells must reach a critical size. The key point in control of the cell cycle is START, the transition that

initiates processes like DNA synthesis in S phase, budding and spindle pole body duplication. Once

cells have passed START, they are irreversibly committed to replicating their DNA and progressing

through the cell cycle. START thus coordinates the cell cycle with cell growth. Nutrient starvation as

well as induction of mating blocks passage through START. There are additional checkpoints that


9/23

arrest cells during the cell cycle to avoid DNA damage or cell death due to events occurring out of

order. These control points are situated at the G1-S and G2-M boundaries and can be considered as

internal regulatory systems that arrest the cell cycle if prerequisites for progression are not met.

After having passed the cell-size dependent START checkpoint, the level of cyclins (Cln,Clb)

dramatically increase. Cyclins are periodically expressed and different cyclins (at least 11 in yeast)

are known to be involved in the control of G1 (G1 cyclins), G2 (B-type cyclins) and DNA synthesis (S

phase cyclins). G1 cyclins are transcriptionally regulated. Cln3p, a particular G1 cyclin, is a putative

sensor of cell size, which acts by modulating the levels of other cyclins. In addition to cyclin

accumulation, the activity of a cylin-dependent kinase (CDK) which is an effector of START, is

induced; this is the gene product of CDC28. Cdc28p (also termed Cdk1p) couples with G1 cyclins that

activate its kinase potential. Homologues of this 34 kDa protein have been characterized in other

eukaryotes. Cdk1 as well as being essential for S phase, is also important in controlling entry into

mitosis. Complex formation of Cdk1 has been established with Cln1-3 (at G1), with Clb5,6 (at S), Clb

3,4 (at S/G2) and Clb1,2 (at M). Alternation of cell cycle phases appears to be due to mechanisms that

one cyclin family succeeds another. The level of cyclins are controlled by synthesis and programmed

proteolysis. In this regard it has been shown that G2 cyclins are necessary for degradation of G1

cyclins, and that G2 cyclin synthesis is coupled to removal of G1 cyclins (Figure 10-8).

Figure 10-8: Regulation of the yeast cell cycle.

Important players in this game are inhibitor proteins, known as CKIs, which block CDK activity in G1.

They represent a key mechanism by which the onset of DNA replication is regulated. One such


10/23

inhibitor is Sic1p: upon its destruction by programmed proteolysis, cyclin-Cdk1 activity is induced. This

degradation then triggers the G1-S transition [Lauren et al., 2001].

In addition to this pathway, cell cycle progression is controlled by the availability of nutrients (Figure

10-9). Nutrient levels (for example, glucose or nitrogenous compounds) regulate the intracellular

concentration of cAMP via a small G protein, Ras. The so-called Ras/cAMP pathway is well

documented (see chapter 13). Decreasing levels lead to G1 arrest, while increasing levels induce the

cAMP-dependent protein kinase (PKA), which then phosphorylates and thereby activates specific

transcription factors involved in START.

Figure 10-9: G1 regulation in yeast.

G2-M control is characterized by the association of Cdk1 with B-type cyclins. Complexing of the kinase

with the cyclins activates the kinase, leading to induction of M phase. At the end of M phase, the

mitotic cyclins are removed by programmed proteolysis.

10.2.2 DNA Replication

Chromosome duplication is central to cell division and is tightly controlled during the cell cycle. In

eukaryotic cells, chromosome duplication is accomplished by initiating replication forks at many origins

of replication on each chromosome; activation of the different replication origins is coordinated

during S phase [Donaldson et al., 1999; Stillman, 2001; Raghuraman et al, 2001 Iyer et al., 2001;

Heun et al., 2001; Wyrick et al., 2001]. ARS elements as substantial elements in yeast replication

origins have been discussed above.


11/23


12/23

Figure 10-11: Regulation of replication by cyclins.

Interestingly, DNA replication must be restricted to only one round in each cell cycle. Work in yeast

has shown that Cdk activity during G2 is required to prevent re-replication of DNA; the mechanism of

this inhibition is not known, only the fact that the Cdks are involved in promoting the degradation of

Cdc6p.

Progression through the cell cycle is highly coordinated. Replication origin firing during S phase is not

random but rather is under strict temporal and spatial control. Replication forks cluster in discrete

'replication factories' within the nucleus and components required for elongation associate with nuclear

structural components such as the lamina. Definitely, early and late origins have to be distinguished.Factors that share responsibility for promoting S phase are two B type cyclins, Clb5p and Clb6p, in

conjunction with a single cyclin-dependent kinase, Cdc28p. As it apperas, Clb5p is executing the origin

firing programme in both early and late origins, while Clb6-Cdc28 can only fire early replication origins.

Further, the origin-firing programme is subject to checkpoint controls (Figure 10-11). One of the

essential players is Rad53p, which is involved in monitoring successful execution of the programme of

DNA replication during S phase, and co-ordinating a controlled arrest if problems are encountered.

Rad53p also seems to be required for maintaining the level of nucleotides in the normal S phase.

10.2.3 Spindle Dynamics

In S. cerevisiae, the mitotic spindle must orient along the cell polarity axis, defined by the site of bud

emergence, to ensure correct nuclear division between the mother and daughter cells [Segal and


13/23

Bloom, 2001]. Establishment of spindle polarity dictates this process and relies on the concerted

control of spindle pole function and a precise programme of cues originating from the cell cortex that

directs the cytoplasmic microtubule attachments during spindle morphogenesis. This cues cross talk

with the machinery responsible for bud site selection, indicating that orientation of the spindle is

mechanistically coupled to the definition of a polarity axis and the division plane.

Spindle morphogenesis in yeast is initiated by the execution of START at the G1-S transition of the

cell cycle. Progression through START triggers bud emergence, DNA replication and the duplication of

the microtubule-organizing centre (MTOC) - the spindle pole body (SPB) (Figures 10-5 and 10-12).

The single stages of mitosis and intracellular movements can be distinguished by time-lapse phase-

contrast microscopy. In addition to the polymerization and depolymerization of tubulin (the major

microtubular protein), cytolasmic dynein is a mechanochemical enzyme or motor protein which drives

microtubules motility in yeast. Actin filaments, either as cytoskeletal cables or as cortical membrane

patches, undergo dynamic changes during the cell cycle. The microtubules emanate from the SPBs

toward the new bud and orientate the nucleus and intranuclear spindle at mitosis. The nuclear

membrane remains intact throughout mitosis with the mitotic spindle forming intranuclearly between

two SPBs embedded in the nuclear envelope. Once the genome replicates, the spindle aligns parallel

to the mother bud axis and elongates eventually to provide each cell with one nucleus.

The program for the establishment of spindle polarity, primed by cellular factors partioning

asymmetrically between the bud and the mother cortex, coupling of this process to bud site selection

and polarized growth has been elucidated in some detail [Segal and Bloom, 2001]. Several cortical

components implicated in spindle orientation such as Bni1p, a target of the polarizing machinery

essential in bud site selection and spindle orientation, and the actin interactor Aip3p/Bud6p are initially

localized to the bud tip. Other cortical elements (e.g. Num1p) are restricted initially to the mother cell

during spindle assembly.

Figure 10-12: Spindle dynamics.


14/23

Figure 10-13: Fluorescence imaging of microtubules.

Factors mediating the process of microtubule attachment with the bud cell cortex are Bim1p and

Kar9p. Bim1p can directly bind to microtubules and is required for the high dynamic instability of

microtubules that is characteristic of cells before spindle assembly. Kar9p has been implicated in the

orientation of functional microtubule attachments into the bud during vegetative growth. It is deliveredto the bud by a Myo2-dependent mechanism presumably tracking on actin cables. Interaction of the

two factors, Bim1p and Kar9p, appears to provide a functional linkage between the actin and

microtubule cytoskeletons. In addition, Bud3p, a protein for axial budding of haploid cells, accumulates

at the bud neck and is required for the efficient association of Bud6p to the neck region. Further, a

variety of motor proteins are necessary in spindle morphogenesis: dynein and the kinesin-like proteins

Kip2p and Kip3p, as well as Kar3p are involved in regulating microtubule dynamics, mediating nuclear

migration to the bud neck and facilitating spindle translocation (Figure 10-13).

10.2.4 Sister Chromatid Cohesion and Separation

Sister chromatid cohesion is essential for accurate chromosome segregation during the cell cycle

[Nasmyth, 1999; Biggins & Murray, 1999; Robert et al.; Nasmyth, 2002; Carnobel & Cohen-Fix; 2002;

Uhlmann, 2004]. A number of structural proteins are required for sister chromatid cohesion and there

seems be a link in some organisms between the processes of cohesion and condensation. Likewise, a

number of proteins that induce and regulate the separation of sister chromatids have been identified.

Chromosome splitting is an irreversible event and must therefore be highly regulated. Once sister

chromatids separate from one another, damage to the genome cannot easily be repaired by

recombination nor can mistakes in chromosome alignment be corrected. Sister chromatids are pulled

to opposite 'halves' of the cell by microtubules that emanate from opposite spindle poles. Thesemicrotubules interdigitate and keep the two poles apart. Subsequently, a second set of microtubules

attaches to chromosomes through specialized 'kinetochores' and pulls them to the poles. In this way,

sister chromatides separate and start to move into opposing poles (Figure 10-14).

However, chromosomes do not remain inactive at this process: cohesion between sister chromatids

generates the tension by which cells align them on the metaphase plate. Cohesion also prevents

chromosomes falling apart because of double-stranded breaks and facilitates their repair by

recombination.


15/23

Figure 10-14: Cohesins: the glue between sister chromatids.

Figure 10-15: Sister chromatid separation.

Cohesin rings. The detection of chromosomal proteins that are essential for sister-chromatid

cohesion during G2 and M phases and subsequent analyses have shown that the molecular basis for

sister chromatid cohesion is a chromosomal protein complex, called cohesin. This complex consists of

at least four subunits which together form a large (>50nm diameter) proteinaceous ring: a pair of

structurally similar members of the SMC (structural maintenance of chromosomes) family, Smc1 and

Smt3; and Scc1 and Ssc3; all of which are encoded by essential genes. The establishment of the

cohesin complex is mediated by Eco1/Ctf7 (in S phase) and Pds5, and may also depend on Ssc2 and

Scc4.


16/23

The cohesin complex is shown in Figure 10-14. The circumference of the cohesin ring largely consists

of flexible coiled-coil of the Smc1 and Smc3 subunits, binding each other in head to head and tail to

tail orientation. The tails are held together by the hinge domains binding each other with high affinity.

The Smc1 and Smc3 heads consist of ABC-type ATPase domains that dimerise with each other after

binding ATP. Further, Scc1 associates with the Smc heads, whereby its N-terminus binds to Smc3 and

its C-terminus to Smc1. This bridge could stabilize the ring structure. ATP hydrolysis weakens the

interactions between Smc1 and Smc3 heads, and any remaining link between the heads has to rely

on Ssc1. Evidence has been accumulating that the cohesin complex binds tightly to chromatin by

encircling and topological trapping. A current model for the trapping mechanism is that ATP hydrolysis

of the ABC-like ATPase domains by cohesin induces a change in the interaction of the Smc heads,

thus coupling potential conformational changes in the Smc proteins to active transport of chromatin

into the ring.

It has to be noted here that it is the Scc1 subunit of cohesin that is cleaved by separase at anaphase

onset to relieve sister chromatid cohesion. Separase is tightly regulated: for the rest of the cell cycle, it

is inactive by being bound to an inhibitory chaperone, called securin, whose destruction during the

spindle checkpoint (see below) takes place only after all chromatid pairs have been aligned correctly

on the mitotic spindle.

Condensin. The cohesin binding to chromatin has similarities with chromatin binding to condensin.

Condensin, a 13 S complex, consists of two Smc proteins, Smc2p and Smc4p, and contains three

other essential subunits, one of which is homologous to Scc1p. Just like cohesin, the topological

structure of condensin is a ring. Condensin associates with chromatin independently of ATP, but ATP

hydrolysis is needed for the binding reaction. A particular feat of condensin is that chromatin wraps

around condensin, generating a torsion in the DNA. Thus it is amenable of contributing to

chromosome compaction. In fact, after partial removal of cohesin rings by the separase reaction,

condensin replaces cohesin.

Sister chromatid separation. Segregation of chromosomes must be a tightly regulated process

(Figure 10-15). The fraction of cohesin that persists on chromosomes until metaphase is responsible

for holding sisters together while they bi-orient during prometaphase. It is an absolute requirement that

the sister chromatids adopt an amphitelic orientation, i.e. that the spindle apparatus can be activated

in a way that allows the traction of sister chromatids to opposite poles of the cell by the microtubules to

occur. (Remember that in yeast microtubules connect kinetochores to spindle poles throughout the cell

cycle).

Two phases can be distinguished: (i) cohesins dissociation from and condensins association with

chromosomes occurs in the complete absence of microtubules yet is capable of separating sister

chromatids to ~0.5 m. This first step in the individualisation process is triggered by the participation of

PLK (Polo-like kinase) which will phosphorylate cohesin (and possibly some other proteins). The

second step, orienting sister chromatids on the mitotic spindle (Bi-orientation) and attaching

microtubules to sister kinetochores, whereby chromosomes come under tension, is promoted by the

Aurora-like kinase Ipl1p and controlled by the spindle checkpoint (see below). (ii) The molecular basis

for sister chromatid separation in the second phase arose from the discovery of mitotic cyclins whose

abundance fluctuates during the cell cycle. As already discussed, mitotic cyclins are regulatory

subunits of a cyclin-dependent kinase, Cdk1p, whose activation triggers mitosis into late G2 phase.

The sudden degradation of cyclin B as cells enter anaphase has an important role in abolishing Cdk1


17/23

activity but is not required for the separation of sister chromatids. The apparatus responsible for

targeting cyclin B degradation is a highly conserved multisubunit particle, called the anaphase

promoting complex (APC/C), which possesses ubiquitin-protein-ligase activty. Ubiquitination

mediated by APC/C requires rate-limiting activator proteins that bind to APC; in yeast these are at

least two proteins, namely Cdc20p and Cdh1p. These activators specify both substrate specificity and

the timing of proteolysis.

In the absence of APC/C function, yeast cells arrest in metaphase, and sister chromatids fail to

segregate owing to the persistence of securin, the inhibitor of separase. Securin accumulates within

nuclei during late G1 phase, is maintained during G2 and early M phase, but degraded shortly before

anaphase, so that separase becomes active. Separase resides in the cytoplasm until cells enter

mitosis, whereupon it accumulates at the mitotic spindle until late anaphase. Separase is activated by

proteolysis of securin by APC/C.

10.2.5 Spindle Checkpoint

Most eukaryotic cells posses a surveillance mechanism, also called spindle checkpoint, that

prevents sister chromatid separation when spindles are damaged or chromosomes fail to form spindle

attachments [Amon, 1999; Nasmyth, 2002]. The kinetochore of a single lagging chromosome emits a

signal capable of blocking separation of all sister pairs. It also blocks any further cell cycle

progression. In yeast, this process is triggered by the production of a complex, which contains the

proteins Mad1p, Mad2p, Mad3p, and the protein kinases Bub1p and Bub3p (which are essential,

integral components of the kinetochore). Bub3p binds to the activator Cdc20p of the APC/C complex

and thereby blocks ubiquitination of both securin and cyclin B. In addition, protein kinase Msp1p is

also required for spindle pole duplication, and the subunit Ncd10p of the centromere binding complex

CBF3 are needed. The Mad complex (Figure 10-16) has been shown to be highly conserved among

other eukaryotes.

Figure 10-16: The spindle checkpoint.


18/23

10.3 Sexual Reproduction

Though vegetative growth is the major way of yeast reproduction, sexual reproduction is an

alternative when nutrient supplies fall short. The latter process involves the conjugation of cells of

opposite mating type [Shimoda, 2004]. A heterokaryon with a diploid set of chromosomes is formed,

which is capable of reproduction by budding. Under starvation conditions, meiosis is induced which

leads to sporulation and finally to the propagation of four haploid spores that segregate 2:2 (Figure 10-

17).

Figure 10-17: Life cycle of S. cerevisiae.

10.3.1 Mating Types and Mating

Cells of opposite mating type (a and ) synchronize each others' cell cycles at START in response to

their mating factors. Once cells progress through START, they are unable to mate until the next cell

cycle. Conjugation occurs by surface contact of specialized projections ('schmoo' formation) on mating

cells followed by plasma membrane fusion. The mechanism of the mating response will be discussed

in more detail in chapter 13.


19/23

Figure 10-18: Mating type switch.

After spore germination, haploid cells have the capability of undergoing mating type switch which

maximizes the potential of diploidy. Wild-type strains are homothallic and a/ diploids represent the

usual vegetative state of this yeast. Industrial (brewing) yeast strains are generally polyploid and do

not undergo a sexual life cycle (Figure 10-18).

10.3.2 Meiosis and Sporulation

Meiosis is a variation on the theme of mitosis. It produces haploid gametes that contain recombinant

chromosomes, parts of which are derived from one parent and parts of which are derived from theother parent. As in the case of mitosis, meiosis starts with a round of DNA replication in diploid cells,

which produces connected sister chromatids (Figure 10-19). Recombination between homologous

chromatids brings homologues together. During the first meiotic division (meiosis I), sister

kinetochores are treated as a single unit, and homologous kinetochore pairs are attached to and are

pulled towards opposite spindle poles. However, as long as at least one reciprocal exchange has

occurred, cohesion between sister chromatid arms will oppose disjunction of homologues and their

segregation to opposite poles of the cell. Thus, loss of sister-chromatid cohesion along chromosome

arms is essential for chromosome segregation during meiosis I. Meanwhile, however, cohesion

between sister centromeres persists so that it can be later used to align sisters on the meiosis II

metaphase plate. The difference in timing of sister chromatid cohesion loss is therefore a crucialaspect. Similar cohesion proteins as required for mitosis seem to work in meiosis.


20/23

Figure 10-19: Meiosis in yeast.Sporulation, which in yeast is induced at (nitrogen) starvation, is regulated by a specialized MAP

kinase signalling pathway (see chapter 13). In many aspects, starvation is similar to other stress

responses in yeast. Spore formation requires the activation of a large number of genes, several of

which are involved in the biosynthesis of spore walls.


21/23


22/23

Manning, B.D., Snyder, M. Drivers and passengers wanted! the role of kinesin-associated proteins.

Trends Cell.Biol. 10 (2000) 281-289.Mala, J., Nurse, P. Trends Cell Biol.8 (1998) 163-169.

Nasmyth, K. Separating sister chromatids. Trends Biochem. Sci.24 (1999) 98-104.

Nasmyth, K. Segregating sister genomes: the molecular biology of chromosome separation. Science

297 (2002) 559-565.

Ni, L., Snyder, M. A genomic study of the bipolar bud site selection pattern in Saccharomyces

cerevisiae. Mol. Biol.Cell12 (2001) 2147-2170.Ooi, S.L., Shoemaker, D.D., Boeke, J.D. A DNA microarray-based genetic screen for non-homologous

endjoining mutants in Saccharomyces cerevisiae. Science294 (2001) 2552-2556.Raghuraman, M.K., Winzeler, E.A., Collingwood, D., Hunt, S., Wodicka, L., Conway, A., Lockhart,D.J., Davis, R.W., Brewer, B.J., Fangman, W.L. Replication dynamics of the yeast genome. Science

294 (2001) 115-121.Ricchetti, M., Fairhead, C., Dujon, B. Mitochondrial DNA repairs double-strand breaks in yeast

chromosomes. Nature402 (1999) 96-100.Roemer, T., Vallier, L.G., Snyder, M. Selection of polarized growth sites in yeast. Trends Cell Biol. 6(1996) 434-441.

Segal, M., Bloom, K. Control of spindle polarity and orientation in S. cerevisiae. Trends Cell Biol.11

(2001) 160-166.

Sheu, Y.J., Barral, Y., Snyder, M. Polarized growth controls cell shape and bipolar bud site selection in

Saccharomyces cerevisiae. Mol. Cell .Biol. 20 (2000) 5235-5247.Shimoda, C. Forespore membrane assembly in yeast: coordinating SPBs and membrane trafficking. J.Cell Sci. 17 (2004) 389-395.

Stillman, B. DNA replication. Genomic views of genome duplication. Science294 (2001) 2301-2304.Swaroop, M., Wang, Y., Miller, P., Duan, H., Jatkoe, T., Madore, S.J., Sun, Y. Yeast homolog of

human SAG/ROC2/Rbx2/Hrt2 is essential for cell growth, but not for germination: chip profilingimplicates its role in cell cycle regulation. Oncogene19 (2000) 2855-2866.Uhlmann, F. The mechanism of sister chromatid cohesion. Exp. Cell Res. 296 (2004) 80-85.

Vogel, J.,Snyder, M. gamma-tubulin of budding yeast. Curr. Top. Dev. Biol. 49 (2000) 75-104.Vogel, J., Drapkin, B., Oomen, J., Beach, D., Bloom, K., Snyder, M. Phosphorylation of gamma-tubulin

regulates microtubule organization in budding yeast. Dev. Cell.1 (2001) 621-631.Wyrick, J.J., Aparicio, J.Gg., Chen, T., Barnett, J.D., Jennings, E.G., Young, R.A., Bell, S.P., Aparicio,

O.M. Genomewide distribution of ORC and MCM proteins in S. cerevisiae: highresolution mapping of

replication origins. Science294 (2001) 2357-2360.


23/23

Zhu, H., Klemic, J.F., Chang, S., Bertone, P., Casamayor, A., Klemic, K.G., Smith, D., Gerstein, M.,

Reed, M.A., Snyder, M. Analysis of yeast protein kinases using protein chips. Nat. Genet. 26 (2000)

283-289.

cell cyle regulation in yeast

Documents